Diagnostic imaging is widely used in contemporary medicine. It requires that an appropriate intensity of signal from an area of interest is achieved in order to differentiate certain structures from surrounding tissues, regardless of the technique used. As noted by Torchilin (1998) imaging involves the relationship between the three spatial dimensions of the region of interest and a fourth dimension, time, which relates to both the pharmacokinetics of the agent and the period necessary to acquire the image. The physical properties that can be used to create an image include emission or absorption of radiation, nuclear magnetic moments and relaxation, and transmission or reflection of ultrasound. According to the physical principles applied, currently used imaging techniques include γ-scintigraphy (involving the application of γ-emitting radio-active materials); magnetic resonance (MR, phenomenon based on the transition between different energy levels of atomic nuclei under the action of radiofrequency signal); computed tomography (CT, the technique which utilises ionising radiation with the aid of computers to acquire cross-images of the body and three-dimensional images of areas of interest); and ultra-sonography (US, the technique using irradiation with ultrasound and based on the different rate at which ultrasound passes through various tissues). All four imaging techniques differ in their physical principles, sensitivity, resolution (both spatial and temporal), ability to provide images without contrast agent-mediated enhancement, and some other parameters, such as cost and safety. Usually, the imaging of different organs and tissues for early detection and localisation of numerous pathologies cannot be successfully achieved without appropriate contrast agents (see further) in different imaging procedures.
To improve imaging contrast agents are used. These are the substances which are able to absorb certain types of signal (irradiation) much stronger than surrounding tissues. The contrast agents are specific for each imaging technique (see Table 1), and as a result of their accumulation in certain sites of interest, those sites may be easily visualised when the appropriate imaging technique is applied.
TABLE 1Imaging techniques and required concentration of diagnostic moietiesRequiredconcentra-Imaging techniqueDiagnostic moietytionGamma-scintigraphyDiagnostic radionuclides, such10−10 Mas 111In, 99mTc, 67GaMagnetic resonance (MR)Paramagnetic ions, such as Gd10−4 Mimagingand Mn, and iron oxideComputed tomography (CT)Iodine, bromine, barium10−2 MimagingUltrasonographyGas (air, argon, nitrogen)from: Torchilin (1998)
The tissue concentration that must be achieved for successful imaging varies between techniques and diagnostic moieties, see table 1. In many cases, contrast agent-mediated imaging is based on the ability of some tissues (i.e., macrophage-rich tissues) to absorb the particulate substances. This process is particle size-dependent and relies on a fine balance between particles small enough to enter the blood or lymphatic capillaries, yet large enough to be retained within the tissue. In any of imaging techniques, two main routes of administration of contrast agent are used: systemic and via local circulation. Each has its own advantages and disadvantages. By varying the physico-chemical properties of a contrast, or contrast carrier, the rate of its disappearance from the injection site upon local administration can be modulated. A disadvantage of systemic administration is that it increases the exposure of non-target organs to potentially toxic contrast agent.
Liposomes
To facilitate the accumulation of contrast in the required zone, various micropartic-ulates have been suggested as carriers for contrast agents. Among those carriers, liposomes, microscopic artificial phospholipid vesicles, draw special attention because of their easily controlled properties and good pharmacological characteristics. Many individual lipids and their mixtures, when suspended in an aqueous phase, spontaneously form bilayered structures (liposomes) in which the hydrophobic parts of their molecules face inwards and the hydrophilic parts are exposed to the aqueous phase surrounding them. Several different types of liposomes exist; each type has specific characteristics and can be prepared by specific methods. Usual classification of liposomes is based on their size and number of concentric bilayers forming a single vesicle (such as multilamellar vesicles (MLV), unilamellar vesicles, LUV, small unilamellar vesicles (SUV)). The methods for producing LUVs can be easily scaled up and used for industrial production of large batches of liposomes with a predictable size and a narrow size distribution.
For almost two decades liposomes have been recognized as promising carriers for drugs and diagnostic agents for the following reasons: (1) Liposomes are completely biocompatible; (2) they can entrap practically any drug or diagnostic agent into either their internal water compartment or into the membrane itself depending on the physico-chemical properties of the drug; (3) liposome-incorporated pharmaceuticals are protected from the inactivating effect of external conditions, yet at the same time do not cause undesirable sidereactions; (4) liposomes also provide a unique opportunity to deliver pharmaceuticals into cells or even inside individual cellular compartments. Pursuing different in vivo delivery purposes, the size, charge and surface properties of liposomes can be easily changed simply by adding new ingredients to the lipid mixture before liposome preparation and/or by variation of preparation methods.
Unfortunately, phospholipid liposomes, if introduced into the circulation, are very rapidly (usual half-clearance time is within 30 min) sequestered by the cells of the reticuloendothelial system (RES). Liver cells are primarily responsible, and the sequestration is relatively dependent on their size, charge, and composition of the liposomes. Circulating peripheral blood monocytes can also endocytose liposomes and later infiltrate tissues and deliver endocytosed liposomes to certain pathological areas in the body.
Until recently, the potential of liposomes as drug carriers has been limited by the rapid clearance of liposomes from the bloodstream. For example, conventional liposomes may be largely cleared from the bloodstream within 1-2 hours after intravenous administration
A variety of approaches for extending the circulation time of liposomes have been proposed. Two of these have been successful in extending the half-life of liposomes in the bloodstream by periods of up to 40-50 hours. In one approach, described in U.S. Pat. No. 4,837,028, liposomes are formulated with the ganglioside GM1 and predominantly rigid lipids. In another general approach, disclosed in U.S. Pat. No. 5,013,556, liposomes are coated with a layer of polyethylene glycol (PEG) chains.
The imaging of the most macrophage-rich organs of RES, liver and spleen, was the earliest one performed with contrast-loaded liposomes, as RES organs are the natural targets for liposomes and accumulate them well upon intravenous administration. The diagnostic imaging of liver and spleen is usually aimed at discovering tumors and metastases in those organs, as well as certain blood flow irregularities and inflammatory processes. The use of at least three different imaging techniques for this purpose was described, namely, γ-, MR-, and CT-imaging.
Tumor Imaging with Contrast Liposomes
One area of important potential application of contrast-loaded liposomes is tumor imaging. The main mechanism of liposome accumulation in tumors is via extravas-ation through leaky tumor capillaries into the interstitial space. As in many other cases, the efficacy of such accumulation can be sharply increased by using long-circulating PEG-coated liposomes. Liposome-based imaging agents have already been successfully used for γ-, MR-, CT-, and sonographic imaging of tumors. Indium 111 labeled liposomes for tumor imaging (VesCan®, Vestar, Inc.) are already in Phase II-III clinical trials.
Liposome formulations may also be used for visualization of inflammation and infection sites. The use of microparticulate imaging agents for the visualization of infection and inflammation sites is based on the ability of microparticulates to extravasate from the circulation and accumulate in those sites similar to what we already described for tumors and infarcted tissues.
The two major problems connected with the use of liposomes for diagnostic imaging purposes namely low efficacy of contrast liposomes in tumor imaging has often been explained by fast clearance of the liposomes from the blood and their inability to accumulate in the diseased tissue. Both of these obstacles are addressed in the present invention.